Biochemical properties Denaturation The strands of the DNA double helix are held together by hydrogen bonding interactions between the complementary base pairs. Heating DNA in solution easily breaks these hydrogen bonds, allowing the two strands to separate—a process called denaturation or melting. The two strands may reassociate when the solution cools, reforming the starting DNA duplex—a process called renaturation or hybridization. These processes form the basis of many important techniques for manipulating DNA. For example, a short piece of DNA called an oligonucleotide can be used to test whether a very long DNA sequence has the complementary sequence of the oligonucleotide embedded within it. Using hybridization, a single-stranded DNA molecule can capture complementary sequences from any source. Single strands from RNA can also reassociate. DNA and RNA single strands can form hybrid molecules that are even more stable than double-stranded DNA. These molecules form the basis of a technique that is used to purify and characterize messenger RNA (mRNA) molecules corresponding to single genes.
Ultraviolet absorption DNA melting and reassociation can be monitored by measuring the absorption of ultraviolet (UV) light at a wavelength of 260 nanometres (billionths of a metre). When DNA is in a double-stranded conformation, absorption is fairly weak, but when DNA is single-stranded, the unstacking of the bases leads to an enhancement of absorption called hyperchromicity. Therefore, the extent to which DNA is single-stranded or double-stranded can be determined by monitoring UV absorption.
Chemical modification After a DNA molecule has been assembled, it may be chemically modified—sometimes deliberately by special enzymes called DNA methyltransferases and sometimes accidentally by oxidation, ionizing radiation, or the action of chemical carcinogens. DNA can also be cleaved and degraded by enzymes called nucleases.
Methylation Three types of natural methylation have been reported in DNA. Cytosine can be modified either on the ring to form 5-methylcytosine or on the exocyclic amino group to form N4-methylcytosine. Adenine may be modified to form N6-methyladenine. N4-methylcytosine and N6-methyladenine are found only in bacteria and archaea, whereas 5-methylcytosine is widely distributed. Special enzymes called DNA methyltransferases are responsible for this methylation; they recognize specific sequences within the DNA molecule so that only a subset of the bases is modified. Other methylations of the bases or of the deoxyribose are sometimes induced by carcinogens. These usually lead to mispairing of the bases during replication and have to be removed if they are not to become mutagenic.
Natural methylation has many cellular functions. In bacteria and archaea, methylation forms an essential part of the immune system by protecting DNA molecules from fragmentation by restriction endonucleases. In some organisms, methylation helps to eliminate incorrect base sequences introduced during DNA replication. By marking the parental strand with a methyl group, a cellular mechanism known as the mismatch repair system distinguishes between the newly replicated strand where the errors occur and the correct sequence on the template strand. In higher eukaryotes, 5-methylcytosine controls many cellular phenomena by preventing DNA transcription. Methylation is also believed to signal imprinting, a process whereby some genes inherited from one parent are selectively inactivated. Correct methylation may also repress or activate key genes that control embryonic development. On the other hand, 5-methylcytosine is potentially mutagenic because thymine produced during the methylation process converts C:G pairs to T:A pairs. In mammals, methylation takes place selectively within the dinucleotide sequence CG—a rare sequence, presumably because it has been lost by mutation. In many cancers, mutations are found in key genes at CG dinucleotides.
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Nucleases Nucleases are enzymes that hydrolytically cleave the phosphodiester backbone of DNA. Endonucleases cleave in the middle of chains, while exonucleases operate selectively by degrading from the end of the chain. Nucleases that act on both single- and double-stranded DNA are known.
Restriction endonucleases are a special class that recognize and cleave specific sequences in DNA. Type II restriction endonucleases always cleave at or near their recognition sites. They produce small, well-defined fragments of DNA that help to characterize genes and genomes and that produce recombinant DNAs. Fragments of DNA produced by restriction endonucleases can be moved from one organism to another. In this way it has been possible to express proteins such as human insulin in bacteria.
Mutation Chemical modification of DNA can lead to mutations in the genetic material. Anions such as bisulfite can deaminate cytosine to form uracil, changing the genetic message by causing C-to-T transitions. Exposure to acid causes the loss of purine residues, though specific enzymes exist in cells to repair these lesions. Exposure to UV light can cause adjacent pyrimidines to dimerize, while oxidative damage from free radicals or strong oxidizing agents can cause a variety of lesions that are mutagenic if not repaired. Halogens such as chlorine and bromine react directly with uracil, adenine, and guanine, giving substituted bases that are often mutagenic. Similarly, nitrous acid reacts with primary amine groups—for example, converting adenosine into inosine—which then leads to changes in base pairing and mutation. Many chemical mutagens, such as chlorinated hydrocarbons and nitrites, owe their toxicity to the production of halides and nitrous acid during their metabolism in the body.
